Influence of genetic co‐mutation on chemotherapeutic outcome in NPM1‐mutated and FLT3‐ITD wild‐type AML patients

Abstract Background Nucleophosmin 1 (NPM1) gene‐mutated acute myeloid leukemia (NPM1 mut AML) is classified as a subtype with a favorable prognosis. However, some patients fail to achieve a complete remission or relapse after intensified chemotherapy. Genetic abnormalities in concomitant mutations contribute to heterogeneous prognosis of NPM1 mut AML patients. Methods In this study, 91 NPM1‐mutated and FLT3‐ITD wild‐type (NPM1 mut /FLT3‐ITD wt) AML patients with intermediate‐risk karyotype were enrolled to analyze the impact of common genetic co‐mutations on chemotherapeutic outcome. Results Our data revealed that TET1/2 (52/91, 57.1%) was the most prevalent co‐mutation in NPM1 mut AML patients, followed by IDH1/2 (36/91, 39.6%), DNMT3A (35/91, 38.5%), myelodysplastic syndrome related genes (MDS‐related genes) (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes) (35/91, 38.5%), FLT3‐TKD (27/91, 29.7%) and GATA2 (13/91, 14.3%) mutations. Patients with TET1/2 mut exhibited significantly worse relapse‐free survival (RFS) (median, 28.7 vs. not reached (NR) months; p = 0.0382) compared to patients with TET1/2 wt, while no significant difference was observed in overall survival (OS) (median, NR vs. NR; p = 0.3035). GATA2 mut subtype was associated with inferior OS (median, 28 vs. NR months; p < 0.0010) and RFS (median, 24 vs. NR months; p = 0.0224) compared to GATA2 wt. By multivariate analysis, GATA2 mut and MDS‐related genesmut were independently associated with worse survival. Conclusion Mutations in TET1/2, GATA2 and MDS‐related genes were found to significantly influence the chemotherapeutic outcome of patients with NPM1 mut AML. The findings of our study have significant clinical implications for identifying patients who have an adverse response to frontline chemotherapy and provide a novel reference for further prognostic stratification of NPM1 mut /FLT3‐ITD wt AML patients.

5][6][7] The most common NPM1 mutation is a 4 base-pair insertion in exon 12 of the C-terminal.The base-pair insertion causes frameshifts and generates a novel C-terminal nuclear export signal, which is responsible for cytoplasmic localization of mutant NPM1 protein, while the wildtype NPM1 predominantly localizes within the nucleolus.The aberrantly localized NPM1 had the capability to inhibit caspase-6/-8 mediated myeloid differentiation.[10][11] NPM1-mutated (NPM1 mut ) AML was recognized as an independent category with a favorable prognosis, based on the recently updated risk stratification by the European LeukemiaNet.Additionally, patients harboring both NPM1 and internal tandem duplication of FLT3 (FLT3-ITD) mutations were classified into intermediate-risk group, irrespective of the allelic ratio. 12The NPM1-mutated and FLT3-ITD wild-type (NPM1 mut /FLT3-ITD wt ) patients had clinically heterogeneity, with approximately 50% of patients died of progressive disease. 4,7,13,14The role of genetics co-mutation was essential in deciphering the heterogeneity of NPM1 mut AML patients.Thus, it is necessary to investigate the correlation between genetic co-mutation and clinical outcome in NPM1 mut AML.
The prognostic impact of certain genetic comutations, including TET1/2, IDH1/2, DNMT3A, FLT3-TKD, GATA2, and MDS-related genes in NPM1 mut AML patients is still not fully understood, and conflicting results have been reported in recent studies. 15We conducted a retrospective study to investigate the influence of commonly coexisting mutations on chemotherapeutic outcomes in patients with intermediate-risk karyotype and NPM1 mut /FLT3-ITD wt AML.Our results demonstrated that GATA2 mut was associated with poor overall survival (OS) and relapse-free survival (RFS), TET1/2 mut was related to inferior RFS but not OS.In multivariable Cox regression analysis, GATA2 mut and MDS-related genes mut were independent poor prognostic markers in NPM1 mut /FLT3-ITD wt AML patients.

| Patients
In this study, newly diagnosed NPM1 mut /FLT3-ITD wt AML patients with intermediate-risk karyotype aged from 18 to 73 years old were enrolled in Nanfang Hospital (Guangzhou, China) from 2017 to 2020. 16The exclusion criteria were as follows: (1) individuals under the age of 18 years, (2) patients with acute promyelocytic leukemia and secondary/ transformed leukemia, (3) other carcinomas or severe organ dysfunction at diagnosis, (4) data is not available.The treatment strategy administered to the patients is illustrated in Figure 1.All patients were followed up 30 months for survival analysis.Patients who have undergone hematopoietic stem cell transplantation (HSCT) were considered lost to follow-up from the date of transplantation.

K E Y W O R D S
acute myeloid leukemia, chemotherapy, molecular genetics, survival | 3 of 12 WU et al.

| Definition of clinical end points
Treatment response was evaluated according to standardized criteria. 12The definition of achieved complete remission with or without complete blood count recovery (CR/ CRi) was the presence of <5% bone marrow blasts, absence of peripheral blasts, and absence of extramedullary disease, with or without complete blood count recovery.Relapsed disease was defined as the presence of bone marrow blasts ≥5%, progression of extramedullary disease or reappearance of peripheral blasts.The OS was calculated from the date of diagnosis until death or last follow-up, with censoring at the last follow-up if no event occurred.
The RFS was measured from the date of remission until either the occurrence of relapse, death from any cause, or last follow-up, with censoring at the last follow-up if no event occurred.MRD was defined as the presence of residual leukemia cells below the limit of detection through conventional morphologic evaluation after achieving complete remission.

| Statistical analyses
The analysis of categorical variables was conducted using either Pearson's chi-squared test or Fisher's exact test.The F I G U R E 1 Treatment strategy of the patients.Patients received induction chemotherapy based on "3 + 7" regimen (idarubicin 10 mg/m 2 or daunorubicin 60 mg/m 2 , days 1-3; cytarabine 100 mg/ m 2 , days 1-7).The treatment response was assessed through bone marrow morphology 14-21 days after induction.Patients who achieved complete remission (CR/CRi) after induction received two cycles of consolidation chemotherapy based on high dose of cytarabine (HD-Ara-C 2 g/m 2 , days 1-3).Those without CR/CRi received re-induction chemotherapy (HD-Ara-C 2 g/m 2 plus cladribine 5 mg/m 2 , days 1-5 and G-CSF 300 μg, days 0-5).Subsequent treatment strategies were determined based on MRD status after two cycles of consolidation chemotherapy.Patients with negative MRD received two additional cycles of consolidation chemotherapy or auto-HSCT, while those with positive MRD were suggested to undergo allo-HSCT, unless they lacked HLA-matched donors or declined transplantation.
Mann-Whitney U-test or Kruskal-Wallis test was employed for the analysis of continuous variables.Survival data were compared using the log-rank test and Cox regression analysis (stepwise selection procedure).Since transplantation strategies may have influenced clinical outcomes, survival data were analyzed after censoring at the time of transplantation.Statistical analyses were performed using IBM SPSS 27.0 and GraphPad Prism 9. A two-sided p-value of <0.05 was considered to indicate statistical significance.
There was no significant effect on the rates of CR/CRi, MRD negativity or relapse when the analysis of chemotherapeutic response was performed separately for TET1, TET2, IDH1, and IDH2 (Tables S4 and S5).
Additionally, the survival effect of IDH1 and IDH2 was analyzed separately and did not show statistical significance.Although patients with TET1 mut /TET2 mut had a shorter RFS than those with TET wt (median, NR vs. NR; p = 0.0272), the sample size of TET1 mut /TET2 mut was small and only provided reference value (Figure S1).
Cox regression analysis was conducted to assess the impact of sex, age, white blood count, platelet count, hemoglobin level, lactate dehydrogenase level, bone marrow blasts percentages, peripheral blasts percentages, abnormal karyotype, and co-mutation status on OS and RFS (Tables S7 and S8).The results revealed that patients with GATA2 mut had a significantly higher risk of death with a hazard ratio (HR) of 25.573 (95% CI 4.136-158.124),while those with MDS-related genes mut had an increased risk of death with an HR of 8.366 (95% CI 1.302-53.748).Patients with GATA2 mut had a 3.421fold increased hazard of recurrence (HR, 3.421; 95% CI 1.114-10.508)(Figure 6).

F I G U R E 4
Kaplan-Meier survival analysis of genetic mutation versus corresponding non-mutaion groups in NPM1 mut patients.(A, B) Kaplan-Meier curves comparing TET1/2 mut and TET1/2 wt showed a significant difference in RFS, but no significant difference was observed in OS. (C-F) IDH1/2 mut and DNMT3A mut had no significant impact on OS and RFS.
The existing research findings suggest that NPM1 mut AML is a subtype characterized by genetic and clinical heterogeneity, rather than being a homogeneous entity.The investigation of multiple concomitant mutations has significant implications on formulating an optimal therapeutic strategy.This study implied that mutations in TET1/2, GATA2 and MDS-related genes should be considered when assessing chemotherapeutic outcome in NPM1 mut AML patients.
0] The slight difference may be attributed to the exclusion of FLT3-ITD mut patients.The limitation of our cohort to patients with intermediate-risk karyotypes may constitute additional factor.Although a favorable prognosis was a feature common to the majority of patients with NPM1 mut AML, poor clinical outcomes were observed in some patients, which did seem to be related to the presence of these co-mutations.We noted that the effect of TET1/2, GATA2, and MDS-related genes mutations on chemotherapeutic outcome was prominent.Notably, we observed a significant difference in RFS between patients with TET1/2 mut and TET1/2 wt , but not in OS.The fact that a proportion of relapsed patients were still alive at the end of follow-up probably accounts for this.The relatively limited duration of the follow-up may potentially obscure the effects of TET1/2 mut on OS.Remarkably, contrary to the findings of multivariable Cox regression analysis, the results of univariate analysis did not indicate survival difference in MDS-related genes, even within subgroups characterized by normal karyotype or individuals below 60 years old.The impact of MDS-related genes on survival is revealed when controlling for covariates.It cannot be ruled out that there may exist potential confounding factors, which were not accounted for in this study.Furthermore, considering the unique composition of clustered multiple genes associated with MDS, it is imperative to investigate whether patients within this group can be defined as a homogeneous cohort.
There have been conflicting conclusions regarding whether these genes are independently associated with survival in NPM1 mut AML.1][32][33][34][35] The adverse survival events in NPM1 mut AML group may not be solely explained by the double-mutant type.7][38] It emphasizes the criticality of exploring wider genomic context to constitute a more robust strategy for prognostic stratification.
The pathogenic dysfunction of GATA2 gene is increasingly gaining significance in clinical management of leukemia.0][41] The GATA2 mutation was classified as a clonal hematopoiesis of oncogenic potential (CHOP-like) mutation, which predicts an adverse prognosis when it persists or is acquired post-remission. 424][45] However, given the infrequent occurrence of GATA2 mutation 4,19 there is a lack of studies investigating its survival effect in the NPM1 mut subgroup.Our data uncover a previously unrecognized role of GATA2 mutation as a poor prognostic marker in NPM1 mut AML.The relevant studies have reported that mutant NPM1 increased the expression of GATA2 through bounding to the −77 kb enhancer region of GATA2. 11A Speculative interpretation is that mutant NPM1 may disturb activation of GATA2mediated transcription in NPM1 mut /GATA2 mut AML.
In contrast to our findings, a recent study indicated that patients with DNMT3A mut did not exert a significant impact on CR rate. 337] This may explain why cases with DNMT3A mut showed a higher rate of CR/CRi in our cohort.Monitoring the MRD level has an important value in prognostic evaluation and therapeutic stratification.FCM was used to monitor MRD in F I G U R E 6 Multivariable Cox regression analysis of OS and RFS in NPM1 mut patients (n = 91).Sex, age, WBC count, platelet count, hemoglobin level, LDH level, BM blasts percentages, PB blasts percentages, abnormal karyotype, and co-mutation status (TET1/2, IDH1/2, DNMT3A, MDS-related genes, FLT3-TKD, and GATA2 mutations) were analyzed by univariate Cox regression analysis.Variables with a p-value of less than 0.5 in univariate Cox-regression analysis were selected for inclusion in the multivariable Cox regression analysis.The optimal combination of covariates for multivariable Cox regression was determined via a stepwise selection procedure.
this study.2][53] Therefore, our data may not fully represent the impact of concomitant mutations on MRD.The combination of FCM and molecular detection exhibits enhanced efficiency in evaluating MRD levels.
Although this study has significant implications for precision treatment, the conclusion drawn from the retrospective study should be interpreted with caution.Further research, including multicenter studies with larger samples and prospective studies, is needed to validate these observations.Unfortunately, the availability of both germline and remission samples was limited in this study.Therefore, we could not determine whether these mutations were somatic or germline.
In summary, our retrospective study confirmed that TET1/2, GATA2 and MDS-related genes mutations were related to poor therapeutic outcomes in patients with NPM1 mut AML.These findings provided valuable insights for refining risk stratification, improving prognosis assessment, and optimizing treatment strategies in individuals with NPM1 mut AML.

F I G U R E 2
Mutational landscape of patients with NPM1 mut at diagnosis.Each column represents an individual case, and each row represents a single gene.Red for mutants, and blue for wild-types.Mutation in the bZIP region of the CEBPA gene was not detected in patients with CEBPA sm .SF3B1 was not detected in all cases.F I G U R E 3 Distribution of CR/CRi, MRD-negativity and relapse in NPM1 mut patients based on co-mutation status.(A) Patients harboring DNMT3A mut had a significantly higher percentage of CR/CRi after the first cycle of induction chemotherapy.(B) The impact of achieving CR/CRi after 1-2 cycles of induction chemotherapy did not differ by co-mutation status.(C-E) Co-mutations status had no influence on the rate of MRD-negativity and relapse.

F I G U R E 5
Kaplan-Meier survival analysis of genetic mutation versus corresponding non-mutaion groups in NPM1 mut patients.(A-D) MDS-related genes mut and FLT3-TKD mut had no significant influence on OS and RFS.(E, F) Kaplan-Meier curves comparing GATA2 mut and GATA2 wt showed a significant difference in OS and RFS.
Clinical characteristics of the NPM1 mut patients.: BM, bone marrow; CR/CRi 1 , percentage of CR/CRi post the first cycle of induction chemotherapy; CR/CRi 2 , percentage of CR/CRi post the 1-2 cycles of induction chemotherapy; LDH, lactate dehydrogenase; MRD-1 , percentage of MRD negativity post the 1-2 cycles of induction chemotherapy; MRD-2 , percentage of MRD negativity post the first cycle of consolidation chemotherapy; PB, peripheral blood; WBC, white blood cell.